The nucleus controls gene expression patterns that characterize the function of individual cells. Responses that occur in the nucleus, such as transcription and splicing of pre-mRNA, are elaborate processes that utilize many factors. Because platelets circulate without a nucleus, they have been stereotyped as a cell that lacks complexity and is devoid of nuclear functions. However, our group has recently shown that platelets carry a functional spliceosome and in response to activating signals, platelets splice pre- mRNA into mature, translatable messages. This result was unexpected because pre- mRNA splicing was previously thought to reside only in the nucleus. Possession of a functional spliceosome demonstrates remarkable specialization and intricate information transfer from parental megakaryocytes to terminally differentiated platelets. It also raises the possibility that megakaryocytes endow platelets with other novel gene expression pathways during thrombopoiesis. Recent evidence indicates that the long interspersed nuclear element (LINE-1;also referred to as L1 elements) family of retrotransposons harbor reverse transcriptase activity that regulates cellular growth, differentiation and gene expression. This suggests that L1 elements may regulate megakaryopoiesis and proplatelet formation. If transferred to proplatelets, L1 elements may also control functional responses in mature, circulating platelets. Whether L1 elements control megakaryocyte and platelet function, however, has not been tested. Therefore, in this R21 application we will explore the possibility that megakaryocytes and platelets possess functional L1 elements. In specific aim 1 we will characterize the L1 machinery in megakaryocytes. We will determine if L1 encoded proteins are present in human stem cell-derived megakaryocytes and if they regulate megakaryocyte differentiation and proplatelet formation. In aim 2 we will characterize the L1 machinery in mature, human platelets. We will determine if L1 elements are present in platelets and if they possess reverse transcriptase activity. We also determine if L1 elements control previously-unrecognized functions of platelets that include insertion of L1 sequences into mitochondrial DNA and reverse transcription of mRNA into cDNA. Studies in aim 2 will examine responses in freshly-isolated platelets and in stored platelets that would otherwise be used for platelet transfusions. Together these studies will provide novel insight into the cellular activities of human megakaryocytes and platelets. Studies in aim 2 will also provide important clinical information, especially in regards to HIV-infected patients who are medicated with reverse transcriptase inhibitors and patients who receive platelet transfusions.